DNA quantification by fluorometry (answer to T.Pfitzner)

Oswaldo oswaldo at iats.csic.es
Thu Aug 17 05:24:02 EST 2000


Hi, we had this thread about fluorometry for DNA quantification a few
days ago and someone asked me by private email the question below. Sorry
but I cannot answer privately, there seems to be a problem with the
address.


Thomas Pfitzner asked:
> 
> Hi Oswaldo!
> Do you know, why it is essential to use TNE for Dilution of DNA?


Apparently the Hoechst 33258 dye needs the presence of a minimum amount
of NaCl for full fluorescence. As they say in their technical bulletin,
at least 200mM is necessary when measuring purified DNA but 2.0 to 3.0M
can be used with crude samples. It seems that in these crude samples the
high salt causes dissociation of proteins and DNA thus allowing a
uniform binding of dye to the DNA.

All I know is that I once tried to use TE buffer because I had run
out of TNE, and it did not work. Readings were extremely low and
inconsistent.

If anyone needs the technical bulletin with tips and protocols for DNA
quantification by fluorometry, it is available in pdf format, from
Amersham Pharmacia Biotech site or I can send a copy by email.

Cheers

Oswaldo






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